The concept of antigen specific TCR transduced lymphocytes for adoptive immunotherapy is gaining increasing support. For this to become a reality, however, the biology and in vivo response of these cells to the host environment, including tolerance, antigen presentation, and tumor microenvironment will have to be carefully delineated. Our translational research group recently has developed a series of novel observations/reagents to address this issue. First, we have observed that cyclophosphamide (CTX) preconditioning induces post-lymphopenia expansion of DC in the PBL. Second, we have delineated that IL- 12 conditioning during in vitro priming is able to promote the acquisition of an early effector (TEA) like phenotype. Both are associated with enhanced anti-tumor responses in vivo. Third, our program collaborator (Dr. Shikar Mehrotra) has successfully created a TCR transgenic mouse (termed h3T) which expresses functional melanoma antigen (tyrosinase) specific human TCR in peripheral CD8+ T cells thus providing a source of naive endogenous T cells expressing tyrosinase specific TCR and TCR transduced T cells of the same specificity. We hypothesize that combining the antigen specific response of TCR transduced CD8+ T cells with an environment which promotes DC function will result in the generation of more effective anti-tumor immunity. Therefore, we propose the following: Specific Aim 1 will define the impact of homeostatic proliferation and mechanisms of IL-12 conditioning on TCR transduced T cell survival and function in a tumor bearing host. We will then assess the mechanisms by which IL-12 conditioning impacts on TCR transduced and h3T transgenic T cell survival looking specifically at CTLA-4, PD-1 signaling. Specific Aim 2 will define the TCR transduced CD8+ T cell response to antigen presentation in an environment which promotes DC function evaluating both the post-lymphopenia expansion of DC environment, and also with a limited lymphopenia post CD8+ antibody depletion. PBL from the phase I clinical trial will be used to confirm our preclinical data for both aims. Finally, Specific Aim 3 will determine the optimal conditions required to induce a robust memory TCR transduced CD8+ T cell response in a tumor bearing environment evaluating the mechanisms affecting in vivo TCR transduced CD8+ T cells responses We will then define the therapeutic efficacy of TCR transduced T-cells adoptively transferred into a tumor bearing mouse. Using these novel tools to probe the mechanisms involved in the in vivo response of TCR transduced T cells to the host environment should provide an ability to design more effective adoptive immunotherapy protocols.